Project Summary Humoral immunity relies on B cells to differentiate into plasma cells, which then secrete antibodies. These antibodies bind to antigen to facilitate neutralization and clearance of microbes. Natural infection and vaccination induce differentiation of nave B cells to plasma cells or to memory B cells. Memory B cells, unlike plasma cells, maintain responsiveness to antigen, and can be stimulated by subsequent challenge, similar to a nave B cell. However, upon secondary stimulation, memory B cells differentiate faster to plasma cells and produce higher affinity, more class-switched antibody. Memory B cells can be IgM+ or isotype-switched. It is now appreciated that IgM+ memory B cells are key players in the reactivation response. IgM+ memory B cells respond in a quantitatively and qualitatively more efficient manner than nave B cells, similar to class-switched memory B cells. This suggests that the differences in memory B cell responses are not simply due to class-switched B cell receptors. We propose that memory B cells have an altered chromatin architecture that facilitates their enhanced ability to respond to subsequent infection. These changes are likely in the promoter or enhancer regions of key genes involved in metabolism and plasma cell differentiation, such as Pdhx and Xbp1, respectively. Our genomics data shows that plasma cells rely on more oxidative phosphorylation than nave and activated B cells. Because memory B cells differentiate to plasma cells faster, it suggests that memory B cells will be able to perform oxidative phosphorylation more effectively upon stimulation than nave B cells. We have also shown that naive B cell differentiation to plasma cells is coupled to cell division and to specific epigenetic changes that occur during each division. We do not know whether memory B cells follow the same or similar epigenetic patterns. The overall goal of this proposal is to identify genetic factors that contribute to enhanced reactivation by memory B cells. To accomplish this, Aim 1 will use a variety of infection / inoculation methods to determine the biochemical and epigenetic programs that define metabolism of memory B cells. Aim 2 will characterize the differentiation programs of nave, IgM+ and class-switched memory B cells to plasma cells by seeking to understand the number of divisions required for memory cell differentiation to a plasma cell and how the transcriptome changes during secondary challenge. Thus, the work proposed will define the changes in metabolic gene programming that are mediated by epigenetic changes and enhance our understanding of how memory B cells respond to immune challenge.